Phosphorylation of proteins, one of the most impotent post translational modification (PTM), play impotent roles to control many cellular processes in signal transduction, gene expression, apoptosis. Therefore considerable effort has been devoted to developing methods to detect the phosphoprotein and quantify the phosphorylation states of the protein. In general, phosphorylation of protein, one of post-translational modifications of protein in biological medium, is observed in aminoacid residue such as serine, threonine, histidine, and lysine. Phosphorylation is reversible process, mainly controlled by protein kinase for phosphorylation and by phosphatase for dephosphorylation.
It is widely recognized that irregularity of protein phosphorylation states, such as abnormal phosphorylation or dephosphorylation, relates to nanny diseases. Thus the development of efficient diagnostic method for irregularity of phosphorylation states is very urgent and impotent in the understanding of a variety of biological states relevant to diseases and will contribute to study for diagnose and treatment of diseases.
Mass spectrometry has become an increasingly viable, powerful alternative to old traditional methods for phosphorylation analysis. Nevertheless, analysis of phosphorprotein and phosphopeptide presents great challenges to be solved in mass spectrometry. First, phosphoprotein is often present in substoichiometric amount as a result of partial post-translational modification, and the phosphopeptides digested are present in lower abundance than the other unphosphorylated counterparts in the sample mixture. Second, negatively charged modification, as in phosphoprotein, may be less proteolytic activity by trypsin, and the regions of interest can be missed consequently in mass analysis. Third, the ionization of negatively charged phosphopeptides in a positive mode are also suppressed relative to other unphosphorylated peptides, and the suppression effect is especially increased according to increase of complexity of the peptide mixture. Forth, the partial dephosphorylation of phosphate group from phosphopeptide during mass analysis can be occurred by virtue of chemical instability of phosphate linkage. It can result in decrease of mass sensitivity and increase of complexity of mass spectrum, and become cause of incomplete peptide sequence in mass/mass (MS/MS) sequencing.
For purification, enrichment and high-sensitive mass analysis of phosphopeptides digested from phosphoproteins, several analytical tools were developed such as antibody affinity enrichment, immobilized metal affinity chromatography (IMAC) enrichment, and chemical tagging methods of phosphorylation sites. Among above methods, chemical tagging methods of phosphorylation sites are a efficient analytical tool for phosphoproteome analysis, especially in proteomic field using mass spectrometry. Some chemical tagging methods, based on the b-elimination of phosphor serine (or phosphorthreonine) to generate dehydroalanine (or b-methyldehydroalanine) under basic conditions and the conjugate addition of highly nucleophilic tagging reagent to the resulting conjugate peptides, have been reported for enrichment, MS/MS sequence and relative quantitation of phosphorpeptides digested from phosphorproteins. Reagents having sulfhydryl functionality, such as mercaptoethanol, mercaptoethylamine, ethanedithiol etc., is usually selected as a nucleophile for tagging reaction. The chemical tagging methods especially have merit for high sensitive detection of phosphopeptides, because of the replacement of phosphate group with chemical tag having a high proton affinity increase the ionizability of peptide moiety originally phosphorylated. This chemically tagged peptide is now unsuppressed in mass analysis, and will show greatly increased detectability comparable to that in originally unphosphorylated peptides, even in complex peptide mixture. Additionally the mass shift in spectra due to dephosphorylation (−98 Da) and attachment of chemical tag will help to identify phosphopeptides in complex mass spectra and to assign the sites of phosphorylation in phosphorpeptides.
For high sensitive detection and easy assignment of phosphopeptides from tryptic digest peptides mixture of phosphoprotein, we developed a new β-elimination/chemical tagging method using tag reagent containing guanidine moiety. Guanidination of C-terminal lysine residue of tryptic peptide mixture to hanoarginine residue using O-methyl isourea was reported to increase the mass sensitivity of the modified peptides in positive ion made mass analysis. Gianidino group is actually more basic than that of primary amino group. Gianidinoethanethiol (GET) having both guanidine moiety and high nucleophilic sulfhydryl moiety has a proper structure as a tagging reagent for tagging of phosphorylation sites in phosphorpeptides and phosphoproteins. GET is generated from commercially available (lanidinoethyl disulfide (GEDS) by a proper reducing reagent and compatible with aqueous condition for tagging reaction of phosphorpeptides and phosphorproteins.
On the other hand, the chemical tagging method for phosphoproteins can be carried out in phosphoprotein level before enzymatic digestion of phosphoproteins. A variety of chemical tags are developed and applied to tag phosphoproteins. Enzymatic digestion for protein chemically tagged at the sites formerly phosphorylated is affected from the tag moieties in its enzymatic selectivity. Knight, Z. A. et. al.(Nature Biotechnology, Vol. 21, No. 9, p. 1047, September 2003) use aminoethanethiol as a tagging reagent to phosphorylated sites of phosphoprotein. Aminoethylcysteine residue produced from tagging of aminoethanethiol to formerly phosphoserine residue is play like lysine residue in enzymatic digestion using protease such as trypsin. Thus tagging to phosphoprotein using chemical tag inducing phosphospecific proteolysis can be a powerful tool for assigning of phosphorylated sites in phosphoprotein. Peptides resulting from phosphospecific proteolysis will give y1 fragment ion corresponding to aminoethylcysteine fragment in MS/MS peptide sequence analysis. Korea patent application No. 2004-0008046 discloses selective labeling agent for phosphoproteome analysis and phosphorylated site analysis.